This proposal aims to understand the structural basis of pulmonary surfactant's inter-facial activity. Surfactant is the mix of lipids and proteins that coat the alveoli, performing the essential functions of stabilizing the concave air- liquid interface and preventing alveolar collapse at small lung volumes. The ability of surfactant to spread rapidly to form an inter-facial film that can withstand compression during exhalation to very high densities depends upon its mechanical characteristics, which in turn result from its structure. Recently developed microscopic methods now provide direct access to the structural phases in surfactant that previously could be evaluated only indirectly. The investigators have used fluorescence microscopy and Brewster angle microscopy to provide preliminary evidence for three sequential phase transitions during compression of surfactant films: initial separation of a liquid condensed phase containing mostly DPPC from the remaining liquid expanded film; re-mixing of these phases at a critical point; formation of a multi-layered phase with material immediately above and below the inter-facial monolayer. These findings suggest significant modifications of the traditional model for the mechanisms by which surfactant functions in the lung. The proposed studies will test specific hypotheses concerning phase behavior in the surfactant films and the functional significance of these structures. Microscopic methods will determine the phase behavior of surfactant films and the physical characteristics of the different phases. Other experiments will establish the extent to which the different phase behaviors correlate with function in vitro on a captive bubble and in situ in excised lungs. The approach in particular emphasizes the role of individual constituents not just in simple model systems but also in mixtures containing the full complexity of pulmonary surfactant to maximize the physiological significance of the results.